Fluorescent-Antibody Targeting of Insulin-Like Growth Factor-1 Receptor Visualizes Metastatic Human Colon Cancer in Orthotopic Mouse Models

Fluorescent-antibody targeting of metastatic cancer has been demonstrated by our laboratory to enable tumor visualization and effective fluorescence-guided surgery. The goal of the present study was to determine whether insulin-like growth factor-1 receptor (IGF-1R) antibodies, conjugated with bright fluorophores, could enable visualization of metastatic colon cancer in orthotopic nude mouse models. IGF-1R antibody (clone 24–31) was conjugated with 550 nm, 650 nm or PEGylated 650 nm fluorophores. Subcutaneous, orthotopic, and liver metastasis models of colon cancer in nude mice were targeted with the fluorescent IGF-1R antibodies. Western blotting confirmed the expression of IGF-1R in HT-29 and HCT 116 human colon cancer cell lines, both expressing green fluorescent protein (GFP). Labeling with fluorophore-conjugated IGF-1R antibody demonstrated fluorescent foci on the membrane of colon cancer cells. Subcutaneously- and orthotopically-transplanted HT-29-GFP and HCT 116-GFP tumors brightly fluoresced at the longer wavelengths after intravenous administration of fluorescent IGF-1R antibodies. Orthotopically-transplanted HCT 116-GFP tumors were brightly labeled by fluorescent IGF-1R antibodies such that they could be imaged non-invasively at the longer wavelengths. In an experimental liver metastasis model, IGF-1R antibodies conjugated with PEGylated 650 nm fluorophores selectively highlighted the liver metastases, which could then be non-invasively imaged. The IGF-1R fluorescent-antibody labeled liver metastases were very bright compared to the normal liver and the fluorescent-antibody label co-located with green fluorescent protein (GFP) expression of the colon cancer cells. The present study thus demonstrates that fluorophore-conjugated IGF-1R antibodies selectively visualize metastatic colon cancer and have clinical potential for improved diagnosis and fluorescence-guided surgery.     

Near Infra-Red Photoimmunotherapy with Anti-CEA-IR700 Results in Extensive Tumor Lysis and a Significant Decrease in Tumor Burden in Orthotopic Mouse Models of Pancreatic Cancer

Photoimmunotherapy (PIT) of cancer utilizes tumor-specific monoclonal antibodies conjugated to a photosensitizer phthalocyanine dye IR700 which becomes cytotoxic upon irradiation with near infrared light. In this study, we aimed to evaluate the efficacy of PIT on human pancreatic cancer cells in vitro and in vivo in an orthotopic nude mouse model. The binding capacity of anti-CEA antibody to BxPC-3 human pancreatic cancer cells was determined by FACS analysis. An in vitro cytotoxicity assay was used to determine cell death following treatment with PIT. For in vivo determination of PIT efficacy, nude mice were orthotopically implanted with BxPC-3 pancreatic tumors expressing green fluorescent protein (GFP). After tumor engraftment, the mice were divided into two groups: (1) treatment with anti-CEA-IR700 + 690 nm laser and (2) treatment with 690 nm laser only. Anti-CEA-IR700 (100 μg) was administered to group (1) via tail vein injection 24 hours prior to therapy. Tumors were then surgically exposed and treated with phototherapy at an intensity of 150 mW/cm² for 30 minutes. Whole body imaging was done subsequently for 5 weeks using an OV-100 small animal imaging system. Anti-CEA-IR700 antibody bound to the BxPC3 cells to a high degree as shown by FACS analysis. Anti-CEA-IR700 caused extensive cancer cell killing after light activation compared to control cells in cytotoxicity assays. In the orthotopic models of pancreatic cancer, the anti-CEA-IR700 group had significantly smaller tumors than the control after 5 weeks (p<0.001). There was no significant difference in the body weights of mice in the anti-CEA-IR700 and control groups indicating that PIT was well tolerated by the mice.     

Anti-MUC1 antibody inhibits EGF receptor signaling in cancer cells

MUC1 is a type I transmembrane glycoprotein aberrantly overexpressed in various cancer cells. High expression of MUC1 is closely associated with cancer progression and metastasis, leading to poor prognosis. We previously reported that MUC1 is internalized by the binding of the anti-MUC1 antibody, from the cell surface to the intracellular region via the macropinocytotic pathway. Since MUC1 is closely associated with ErbBs, such as EGF receptor (EGFR) in cancer cells, we examined the effect of the anti-MUC1 antibody on EGFR trafficking. Our results show that: (1) anti-MUC1 antibody GP1.4, but not another anti-MUC1 antibody C595, triggered the internalization of EGFR in pancreatic cancer cells; (2) internalization of EGFR by GP1.4 resulted in the inhibition of ERK phosphorylation by EGF stimulation, in a MUC1 dependent manner; (3) inhibition of ERK phosphorylation by GP1.4 resulted in the suppression of proliferation and migration of pancreatic cancer cells. We conclude that the internalization of EGFR by anti-MUC1 antibody GP1.4 inhibits the progression of cancer cells via the inhibition of EGFR signaling.     

The regulatory roles of miRNA and methylation on oncogene and tumor suppressor gene expression in pancreatic cancer cells

Carcinogenesis is driven by an accumulation of mutations and genetic lesions, which leads to activation of oncogenes and inactivation of tumor suppressor genes. However, the molecular mechanisms by which the expression of these genes was regulated in pancreatic cancer remains unclear. In this study, we investigated the regulatory effects of microRNA and methylation on the expression of k-ras, TP53 and PTEN genes in pancreatic cancer cells. The protein and miRNA levels were measured by Western blotting and Northern blotting, respectively. Xenograft pancreatic tumor models were established by inoculating BxPC-1, Capan-2, and Panc-1 tumor cells into athymic nu/nu mice. A disparate level of KRAS, p53, PTEN, Dnmts, and Dicer 1 proteins as well as let-7i, miR-22, miR-143, and miR-29b miRNA was observed in BxPC-1, Capan-2, and Panc-1 cells. Knockdown of Dicer 1 expression in BxPC-3 and Panc-1 cells resulted in significant increases in KRAS, p53, PTEN, and Dnmts protein levels and significant decreases in miR-22, miR-143, let-7i, and miR-29b expression. Knockdown of Dicer 1 expression in Capan-2 cells significantly increased p53 and PTEN expression, while significantly decreased miR-22 and miR-143 expression, but had no effects on PTEN, Dnmts, let-7i, and miR-29b expression. Knockdown of Dicer 1 expression significantly inhibited xenograft BxPC-3 tumor growth, but promoted xenograft Panc-1 tumor growth. In contrast, knockdown of Dicer 1 expression had no effect on xenograft Capan-2 tumor growth. Our study suggested that different pancreatic cancer cell lines exhibited obvious discrepancies in gene expression profiles, implying that different molecular mechanisms are involved in the carcinogenesis of pancreatic cancer subclasses. Our study highlighted the importance of personalized therapy.     

Cell Surface-Associated Anti-MUC1-Derived Signal Peptide Antibodies: Implications for Cancer Diagnostics and Therapy

The MUC1 tumor associated antigen is highly expressed on a range of tumors. Its broad distribution on primary tumors and metastases renders it an attractive target for immunotherapy. After synthesis MUC1 is cleaved, yielding a large soluble extracellular alpha subunit containing the tandem repeats array (TRA) domain specifically bound, via non-covalent interaction, to a smaller beta subunit containing the transmembrane and cytoplasmic domains. Thus far, inconclusive efficacy has been reported for anti-MUC1 antibodies directed against the soluble alpha subunit. Targeting the cell bound beta subunit, may bypass limitations posed by circulating TRA domains. MUC1’s signal peptide (SP) domain promiscuously binds multiple MHC class II and Class I alleles, which upon vaccination, generated robust T-cell immunity against MUC1-positive tumors. This is a first demonstration of non-MHC associated, MUC1 specific, cell surfaces presence for MUC1 SP domain. Polyclonal and monoclonal antibodies generated against MUC1 SP domain specifically bind a large variety of MUC1-positive human solid and haematological tumor cell lines; MUC1-positive bone marrow derived plasma cells obtained from multiple myeloma (MM)-patients, but not MUC1 negative tumors cells, and normal naive primary blood and epithelial cells. Membranal MUC1 SP appears mainly as an independent entity but also co-localized with the full MUC1 molecule. MUC1-SP specific binding in BM-derived plasma cells can assist in selecting patients to be treated with anti-MUC1 SP therapeutic vaccine, ImMucin. A therapeutic potential of the anti-MUC1 SP antibodies was suggested by their ability to support of complement-mediated lysis of MUC1-positive tumor cells but not MUC1 negative tumor cells and normal naive primary epithelial cells. These findings suggest a novel cell surface presence of MUC1 SP domain, a potential therapeutic benefit for anti-MUC1 SP antibodies in MUC1-positive tumors and a selection tool for MM patients to be treated with the anti-MUC1 SP vaccine, ImMucin.     

Monoclonal antibodies recognizing the non-tandem repeat regions of the human mucin MUC4 in pancreatic cancer

The MUC4 mucin is a high molecular weight, membrane-bound, and highly glycosylated protein. It is a multi-domain protein that is putatively cleaved into a large mucin-like subunit (MUC4α) and a C-terminal growth-factor like subunit (MUC4β). MUC4 plays critical roles in physiological and pathological conditions and is aberrantly overexpressed in several cancers, including those of the pancreas, cervix, breast and lung. It is also a potential biomarker for the diagnosis, prognosis and progression of several malignancies. Further, MUC4 plays diverse functional roles in cancer initiation and progression as evident from its involvement in oncogenic transformation, proliferation, inhibition of apoptosis, motility and invasion, and resistance to chemotherapy in human cancer cells. We have previously generated a monoclonal antibody 8G7, which is directed against the TR region of MUC4, and has been extensively used to study the expression of MUC4 in several malignancies. Here, we describe the generation of anti-MUC4 antibodies directed against the non-TR regions of MUC4. Recombinant glutathione-S-transferase (GST)-fused MUC4α fragments, both upstream (MUC4α-N-Ter) and downstream (MUC4α-C-Ter) of the TR domain, were used as immunogens to immunize BALB/c mice. Following cell fusion, hybridomas were screened using the aforementioned recombinant proteins ad lysates from human pancreatic cell lines. Three anti MUC4α-N-Ter and one anti-MUC4α-C-Ter antibodies were characterized by several inmmunoassays including enzyme-linked immunosorbent assay (ELISA), immunoblotting, immunofluorescene, flow cytometry and immunoprecipitation using MUC4 expressing human pancreatic cancer cell lines. The antibodies also reacted with the MUC4 in human pancreatic tumor sections in immunohistochemical analysis. The new domain-specific anti-MUC4 antibodies will serve as important reagents to study the structure-function relationship of MUC4 domains and for the development of MUC4-based diagnostics and therapeutics.     

MUC-1 aptamer targeted superparamagnetic iron oxide nanoparticles for magnetic resonance imaging of pancreatic cancer in vivo and in vitro experiment

This study aims to explore the ability of magnetic resonance imaging (MRI) in mucin 1 (MUC1) modified superparamagnetic iron oxide nanoparticle (SPION) targeting human pancreatic cancer (PC). The MUC1 target-directed probe was prepared through MUC1 conjugated to SPION using the chemical method to assess its physiochemical characteristics, including hydration diameter, surface charge, and magnetic resonance signal. The cytotoxicity of MUC1-USPION was verified by MTS assay. BxPC-3 was cultured with MUC1-USPION and SPION in different concentrations. The combined condition of the targeted probes and cells were observed through Prussian blue staining. The nude mice model of pancreatic cancer was established to investigate the application of the probe. MRI was performed to determine the intensity of the signal of the transplanted tumor, while immunohistochemistry and Western blot analysis were performed to detect the expression of MUC1 after taking the transplanted tumor specimen. The particle size of the prepared molecular probe was 63.5 ± 3.2 nm, and the surface charge was 10.2 mV. Furthermore, the probe solution could significantly reduce the MRI at T₂, and the magnetic resonance transverse relaxation rate (ΔR²) has a linear relationship with the concentration of iron in the solution. The cell viability of MUC1-USPION in different concentrations revealed no statistical difference, according to the MTS assay. In vitro, the MRI demonstrated decreased T2WI signal intensity in both groups, especially the targeting group. In vivo, MUC1 could selectively accumulate in the nude mice model, and significantly reduce the T₂ signal strength. In subsequent experiments, the expression of MUC1 was high in pancreatic cancer tissues, but low in normal pancreatic tissues, as determined by immunohistochemistry and Western blot analysis. The prepared samples can be combined with pancreatic cancer tissue specificity by in vivo imaging, providing reliable early in vivo imaging data for disease diagnosis.     

Soluble MUC1 and serum MUC1-specific antibodies are potential prognostic biomarkers for platinum-resistant ovarian cancer

MUC1 (CA15-3) and MUC16 (CA125) tumor-associated antigens are upregulated in ovarian cancer and can be detected in patients’ sera by standardized tests. We postulated that increased MUC1 and MUC16 antigens augment antibody responses in platinum-resistant ovarian cancer patients and that the frequency and intensity of these responses can be used as immune biomarkers of treatment response and disease outcome. We measured MUC1 and MUC16 tumor expression by immunohistochemistry (IHC), assessed serum antigenic levels and quantitated circulating antibodies by ELISA in a cohort of 28 ovarian cancer patients with platinum-resistant or platinum-refractory ovarian cancer, and treated with intraperitoneal (IP) interleukin-2 (IL-2). MUC1 and MUC16 were overexpressed in tumor samples and showed differential distribution profiles. Serum MUC1 (CA15-3) measurements were elevated in all patients and significantly correlated with increased risk of death (P = 0.003). MUC1-specific IgM and IgG anitbodies were found in 92 and 50% of cases, respectively. Patients with progressive disease had higher mean anti-MUC1 IgG than responders at both early (P = 0.025) and late (P = 0.022) time points during IP IL-2 treatment. Anti-MUC1 IgM antibodies inversely correlated with overall survival at both early (P = 0.052) and late (P = 0.009) time points. In contrast to MUC1, neither soluble MUC16 nor MUC16-specific antibodies were significantly associated with clinical response or overall survival in this study. Increased serum MUC1 and high anti-MUC1 antibody levels are prognostic for poor clinical response and reduced overall survival in platinum-resistant or platinum-refractory ovarian cancer.     

Site-dependent angiogenic cytokine production in human tumor xenografts

Tumor microenvironment plays a critical role in tumor growth, angiogenesis, and metastasis. Differences in site of tumor implantation result in differences in tumor growth, metastasis, as well as response to chemotherapy. We hypothesized that tumor-induced angiogenic growth factor production into the plasma will also be influenced by site of tumor implantation. We evaluated the site-dependent production of angiogenic growth factors in the plasma of tumor bearing animals at two different sites of implantation. Plasma levels of tumor necrosis factor-alpha (TNF-alpha), basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF) were evaluated in nude mice bearing A2780, SKOV-3, or OVCAR-3 human ovarian tumors, as well as Panc-1, AsPC-1, or BxPC-3 human pancreatic tumors grown as subcutaneous (SC) xenografts or in the intraperitoneal (IP) cavity. Plasma VEGF and bFGF levels produced by two ovarian tumor lines and two pancreatic tumor lines were substantially higher when the tumors were implanted in the IP cavity than in the SC space. These studies indicated that the site of tumor implantation was an important determinant in the production of plasma VEGF and bFGF levels. As more and more anti-angiogenic agents are developed, the need for appropriate animal models becomes apparent. These results suggest the demand for an appropriate model for the in vivo evaluation of anti-angiogenesis.     

Diffusion-Weighted MRI Is Insensitive to Changes in the Tumor Microenvironment Induced by Antiangiogenic Therapy

Antiangiogenic treatment (AAT) used in combination with radiation therapy or chemotherapy is a promising strategy for the treatment of several cancer diseases. The vascularity and oxygenation of tumors may be changed significantly by AAT, and consequently, a noninvasive method for monitoring AAT-induced changes in these microenvironmental parameters is needed. The purpose of this study was to evaluate the potential usefulness of diffusion-weighted magnetic resonance imaging (DW-MRI). DW-MRI was conducted with a Bruker Biospec 7.05-T scanner using four diffusion weightings and diffusion sensitization gradients in three orthogonal directions. Maps of the apparent diffusion coefficient (ADC) were calculated by using a monoexponential diffusion model. Two cervical carcinoma xenograft models (BK-12, HL-16) were treated with bevacizumab, and two pancreatic carcinoma xenograft models (BxPC-3, Panc-1) were treated with sunitinib. Pimonidazole and CD31 were used as markers of hypoxia and blood vessels, respectively, and fraction of hypoxic tissue (HF_Pim) and microvascular density (MVD) were quantified by analyzing immunohistochemical preparations. MVD decreased significantly after AAT in BK-12, HL-16, and BxPC-3 tumors, and this decrease was sufficiently large to cause a significant increase in HF_Pim in BK-12 and BxPC-3 tumors. The ADC maps of treated tumors and untreated control tumors were not significantly different in any of these three tumor models, suggesting that the AAT-induced microenvironmental changes were not detectable by DW-MRI. DW-MRI is insensitive to changes in tumor vascularity and oxygenation induced by bevacizumab or sunitinib treatment.     

Mutated SEA-D227A-conjugated antibodies greatly enhance antitumor activity against MUC1-expressing bile duct carcinoma

For the purpose of establishing a new adoptive immunotherapy for bile duct carcinoma (BDC), we have directed our attention to superantigens (SAgs), the most potent known activators of T lymphocytes. In our previous study, staphylococcal enterotoxin A (SEA) was conjugated chemically with MUSE11 mAb, which recognizes the MUC1 cancer-associated antigen, and shown to enhance the specific cytotoxic activity of T-LAK cells against MUC1-expressing BDC cells (TFK-1) in vitro and in vivo. However, it is probable that SEA might cause side-effects because of nonspecific binding to class II positive cells. In order to overcome these, we generated mutated SEA (mSEA) by changing Asp at position 227 of native SEA to Ala, which has reduced affinity to MHC class II molecules, but retains the potential for T cell activation. When mSEA-D227A was administered to rabbits to examine effects on blood pressure, 500 times more mSEA-D227A was tolerated than native SEA. This prompted us to construct a mSEA-D227A-conjugated mAb, reactive with MUC1. It augmented the antitumor activity of T-LAK cells significantly, and furthermore, mSEA-D227A could be conjugated to two bispecific antibodies, BsAb (anti-MUC1 x anti-CD3) and BsAb (anti-MUC1 x anti-CD28), which in combination had greater enhancing effects than mSEA-D227A-conjugated anti-MUC1 mAb, and combination of unconjugated BsAbs. These findings indicate a utility of mSEA-D227A-conjugated antibodies for targeted cancer immunotherapy.     

Evaluation of Efficacy of Radioimmunotherapy with 90Y-Labeled Fully Human Anti-Transferrin Receptor Monoclonal Antibody in Pancreatic Cancer Mouse Models

Objective

Pancreatic cancer is an aggressive tumor and the prognosis remains poor. Therefore, development of more effective therapy is needed. We previously reported that ⁸⁹Zr-labeled TSP-A01, an antibody against transferrin receptor (TfR), is highly accumulated in a pancreatic cancer xenograft, but not in major normal organs. In the present study, we evaluated the efficacy of radioimmunotherapy (RIT) with ⁹⁰Y-TSP-A01 in pancreatic cancer mouse models.

Methods

TfR expression in pancreatic cancer cell lines (AsPC-1, BxPC-3, MIAPaCa-2) was evaluated by immunofluorescence staining. ¹¹¹In-labeled anti-TfR antibodies (TSP-A01, TSP-A02) were evaluated in vitro by cell binding assay with the three cell lines and by competitive inhibition assay with MIAPaCa-2. In vivo biodistribution was evaluated in mice bearing BxPC-3 and MIAPaCa-2 xenografts. Tumor volumes of BxPC-3 and MIAPaCa-2 were sequentially measured after ⁹⁰Y-TSP-A01 injection and histological analysis of tumors was conducted.

Results

MIAPaCa-2 cells showed the highest TfR expression, followed by AsPC-1 and BxPC-3 cells. ¹¹¹In-TSP-A01 and ¹¹¹In-TSP-A02 bound specifically to the three cell lines according to TfR expression. The dissociation constants for TSP-A01, DOTA-TSP-A01, TSP-A02, and DOTA-TSP-A02 were 0.22, 0.28, 0.17, and 0.22 nM, respectively. ¹¹¹In-TSP-A01 was highly accumulated in tumors, especially in MIAPaCa-2, but this was not true of ¹¹¹In-TSP-A02. The absorbed dose for ⁹⁰Y-TSP-A01 was estimated to be 8.3 Gy/MBq to BxPC-3 and 12.4 Gy/MBq to MIAPaCa-2. MIAPaCa-2 tumors treated with 3.7 MBq of ⁹⁰Y-TSP-A01 had almost completely disappeared around 3 weeks after injection and regrowth was not observed. Growth of BxPC-3 tumors was inhibited by 3.7 MBq of ⁹⁰Y-TSP-A01, but the tumor size was not reduced.

Conclusion

⁹⁰Y-TSP-A01 treatment achieved an almost complete response in MIAPaCa-2 tumors, whereas it merely inhibited the growth of BxPC-3 tumors. ⁹⁰Y-TSP-A01 is a promising RIT agent for pancreatic cancer, although further investigation is necessary to improve the efficacy for the radioresistant types like BxPC-3.     

A Versatile Technique for the In Vivo Imaging of Human Tumor Xenografts Using Near-Infrared Fluorochrome-Conjugated Macromolecule Probes

Here, we present a versatile method for detecting human tumor xenografts in vivo, based on the enhanced permeability and retention (EPR) effect, using near-infrared (NIR) fluorochrome-conjugated macromolecule probes. Bovine serum albumin (BSA) and two immunoglobulins—an anti-human leukocyte antigen (HLA) monoclonal antibody and isotype control IgG_2a—were labeled with XenoLight CF770 fluorochrome and used as NIR-conjugated macromolecule probes to study whole-body imaging in a variety of xenotransplantation mouse models. NIR fluorescent signals were observed in subcutaneously transplanted BxPC-3 (human pancreatic cancer) cells and HCT 116 (colorectal cancer) cells within 24 h of NIR-macromolecule probe injection, but the signal from the fluorochrome itself or from the NIR-conjugated small molecule (glycine) injection was not observed. The accuracy of tumor targeting was confirmed by the localization of the NIR-conjugated immunoglobulin within the T-HCT 116 xenograft (in which the orange-red fluorescent protein tdTomato was stably expressed by HCT 116 cells) in the subcutaneous transplantation model. However, there was no significant difference in the NIR signal intensity of the region of interest between the anti-HLA antibody group and the isotype control group in the subcutaneous transplantation model. Therefore, the antibody accumulation within the tumor in vivo is based on the EPR effect. The liver metastasis generated by an intrasplenic injection of T-HCT 116 cells was clearly visualized by the NIR-conjugated anti-HLA probe but not by the orange-red fluorescent signal derived from the tdTomato reporter. This result demonstrated the superiority of the NIR probes over the tdTomato reporter protein at enhancing tissue penetration. In another xenograft model, patient-derived xenografts (PDX) of LC11-JCK (human non-small cell lung cancer) were successfully visualized using the NIR-conjugated macromolecule probe without any genetic modification. These results suggested that NIR-conjugated macromolecule, preferably, anti-HLA antibody probe is a valuable tool for the detection of human tumors in experimental metastasis models using whole-body imaging.     

Internalization of MUC1 by anti-MUC1 antibody from cell membrane through the macropinocytotic pathway

MUC1 is a type I transmembrane glycoprotein aberrantly overexpressed in various cancer cells. It is thought to serve as a physical barrier from the extracellular environment and also as a receptor for various extracellular molecules. However, little is known about the fate of MUC1 during and after the interaction with these molecules. In the present study, we used anti-MUC1 antibody as an interacting molecule and investigated the cellular trafficking of MUC1. Our results showed that: (1) anti-MUC1 antibody was internalized only in MUC1 expressing cells and triggered internalization and down-regulation of MUC1; (2) the internalization of MUC1 by anti-MUC1 antibody required the cytoplasmic tail of MUC1 and was suppressed by inhibitors of Na⁺/H⁺ exchanger, and caveola/raft-dependent internalization, but not by an inhibitor of clathrin-dependent internalization. We conclude that antibody-induced internalization of MUC1 involves the macropinocytotic pathway.     

Dendritic Cells Loaded with Pancreatic Cancer Stem Cells (CSCs) Lysates Induce Antitumor Immune Killing Effect In Vitro

According to the cancer stem cells (CSCs) theory, malignant tumors may be heterogeneous in which a small population of CSCs drive the progression of cancer. Because of their intrinsic abilities, CSCs may survive a variety of treatments and then lead to therapeutic resistance and cancer recurrence. Pancreatic CSCs have been reported to be responsible for the malignant behaviors of pancreatic cancer, including suppression of immune protection. Thus, development of immune strategies to eradicate pancreatic CSCs may be of great value for the treatment of pancreatic cancer. In this study, we enriched pancreatic CSCs by culturing Panc-1 cells under sphere-forming conditions. Panc-1 CSCs expressed low levels of HLA-ABC and CD86, as measured by flow cytometry analysis. We further found that the Panc-1 CSCs modulate immunity by inhibiting lymphocyte proliferation which is promoted by phytohemagglutinin (PHA) and anti-CD3 monoclonal antibodies. The monocyte derived dendritic cells (DCs) were charged with total lysates generated from Panc-1 CSCs obtained from tumor sphere culturing. After co-culturing with lymphocytes at different ratios, the Panc-1 CSCs lysates modified DC effectively promoted lymphocyte proliferation. The activating efficiency reached 72.4% and 74.7% at the ratios of 1∶10 and 1∶20 with lymphocytes. The activated lymphocytes secreted high levels of INF-γ and IL-2, which are strong antitumor cytokines. Moreover, Panc-1 CSCs lysates modified DC induced significant cytotoxic effects of lymphocytes on Panc-1 CSCs and parental Panc-1 cells, respectively, as shown by lactate dehydrogenase (LDH) assay. Our study demonstrates that the development of CSCs-based vaccine is a promising strategy for treating pancreatic cancer.     

Presenilin-2 polymorphisms and risk of sporadic AD: evidence from a meta-analysis

Pancreatic cancer is a malignant neoplasm of the pancreas that usually has a poor prognosis. The investigation of targets that effectively inhibit pancreatic cancer cell proliferation should provide a fundamental basis for the clinical application of gene therapy. Here, high expression levels of ABCC4 protein in thirty-six pancreatic cancer specimens were quantified using an immunohistochemical assay, and the potential of ABCC4 as a therapeutic target for pancreatic cancer was investigated. Inhibition of ABCC4 expression at the mRNA and protein levels was achieved in Panc-1 and BxPC-3 pancreatic cancer cells infected with a lentivirus expressing an ABCC4 short hairpin RNA (shRNA). The downregulation of ABCC4 expression in Panc-1 and BxPC-3 cells significantly inhibited their proliferation and colony formation in vitro, compared to cells infected with mock control (p < 0.05). Moreover, the specific downregulation of ABCC4 led to the accumulation of cells at the G1 phase of the cell cycle. Our findings reveal that the ABCC4 gene promotes pancreatic cancer cell growth and represents a promising target for gene therapy in pancreatic cancer.     

Cytotoxic efficacy of a novel dinuclear platinum(II) complex used with anti-MUC1 in human breast cancer cells

Mucin 1 (MUC1) is overexpressed in various cancer cells especially in breast cancer cells. There are known research works on the use of anti-MUC1 antibody with docetaxel in ovarian cancer, but there are no data about combined therapy platinum compounds with anti-MUC1 in breast cancer. The aim of the study was to evaluate the antiproliferative properties of a new dinuclear platinum(II) complex (Pt12) used with anti-MUC1 in human breast cancer cells. The dinuclear platinum(II) complex (Pt12) has been synthesized, and its cytotoxicity with anti-MUC1 has been tested in both MCF-7 and MDA-MB-231 breast cancer cells. In this study, the effects of Pt12 with anti-MUC1 on collagen and DNA biosynthesis in human breast cancer cells were compared to those evoked by cisplatin and cisplatin with anti-MUC1. The mechanism of action of Pt12 with anti-MUC1 was studied employing flow cytometry assessment of annexin V binding assay. It was found that Pt12 with anti-MUC1 was more active inhibitor of DNA and collagen synthesis as well more cytotoxic agent than Pt12 alone and cisplatin with anti-MUC1. Cytotoxicity of Pt12 with anti-MUC1 against breast cancer cells is due to apoptotic cell death as well as necrotic cell death. These results indicate that the use of Pt12 with anti-MUC1 may constitute a novel strategy in the chemotherapy of breast cancer tumors.     

Targeting Mcl-1 for Radiosensitization of Pancreatic Cancers

In order to identify targets whose inhibition may enhance the efficacy of chemoradiation in pancreatic cancer, we previously conducted an RNAi library screen of 8,800 genes. We identified Mcl-1 (myeloid cell leukemia-1), an anti-apoptotic member of the Bcl-2 family, as a target for sensitizing pancreatic cancer cells to chemoradiation. In the present study we investigated Mcl-1 inhibition by either genetic or pharmacological approaches as a radiosensitizing strategy in pancreatic cancer cells. Mcl-1 depletion by siRNA produced significant radiosensitization in BxPC-3 and Panc-1 cells in association with Caspase-3 activation and PARP cleavage, but only minimal radiosensitization in MiaPaCa-2 cells. We next tested the ability of the recently identified, selective, small molecule inhibitor of Mcl-1, UMI77, to radiosensitize in pancreatic cancer cells. UMI77 caused dissociation of Mcl-1 from the pro-apoptotic protein Bak and produced significant radiosensitization in BxPC-3 and Panc-1 cells, but minimal radiosensitization in MiaPaCa-2 cells. Radiosensitization by UMI77 was associated with Caspase-3 activation and PARP cleavage. Importantly, UMI77 did not radiosensitize normal small intestinal cells. In contrast, ABT-737, an established inhibitor of Bcl-2, Bcl-X_L, and Bcl-w, failed to radiosensitize pancreatic cancer cells suggesting the unique importance of Mcl-1 relative to other Bcl-2 family members to radiation survival in pancreatic cancer cells. Taken together, these results validate Mcl-1 as a target for radiosensitization of pancreatic cancer cells and demonstrate the ability of small molecules which bind the canonical BH3 groove of Mcl-1, causing displacement of Mcl-1 from Bak, to selectively radiosensitize pancreatic cancer cells.